SPINE Volume 39, Number 5, pp 347-355 ©2014, Lippincott Williams & Wilkins
Spine BASIC SCIENCE
Quantitative Study of Parathyroid Hormone (1-34) and Bone Morphogenetic Protein-2 on Spinal Fusion Outcomes in a Rabbit Model of Lumbar Dorsolateral Intertransverse Process Arthrodesis loan A. Lina, BS, Varun Puvanesarajah, BS, Jason A. Liauw, MD, Sheng-fu L. Lo, MD, David R. Santiago-Dieppa, MD, Lee Hwang, MD, Annie Mao, Ali Bydon, MD, Jean-Paul Wolinsky, MD, Daniel M. Sciubba, MD, Ziya Gokaslan, MD, Christina Holmes, PhD, and Timothy F. Witham, MD
Study Design. A posterolateral rabbit spinal fusion model was used to evaluate the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) and teriparatide (PTH [1-341) used individually and in combination on spinal fusion outcomes. O b j e c t i v e . To test the efficacy of parathyroid hormone on improving spinal fusion outcomes when used with BMP-2. S u m m a r y of Background D a t a . Of the more than 250,000 spinal fusion surgical procedures performed each year, 5% to 3 5 % of these will result in pseudarthrosis. Growing controversy on the efficacy and cost of rhBMP-2 for improving spinal fusion outcomes has presented a challenge for clinicians. Research into PTH as an adjunct therapy to rhBMP-2 for spinal fusion has not yet been investigated.
From the Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD. Acknowledgment date: August 8, 2013. First revision date: December 6, 2013. Acceptance date: December 9, 2013. The device(s)/drug(s) that is/are the subject of this manuscript is/are exempt from FDA or corresponding national regulations because: They were used in an animal model where such regulations are not applicable. Current use of recombinant human bone morphogenetic protein-2 in the clinic is FDAapproved for lumbar spinal fusion surgery. This is functionally analogous to the posterolateral intertransverse arthrodesis procedure that is used In our rabbit model. On the contrary, parathyroid hormone (PTH) (1-34), although FDA-approved for the treatment of patients with osteoporosis, has been used in the literature for similar New Zealand White rabbit spinal fusion models. Concurrently, FDA regulations do not apply to this model. The manuscript includes unlabeled/investigational uses of the products/devices listed below and the status of these is disclosed in the manuscript: teriparatide is approved for use in the treatment of osteoporosis. In this study, we investigate its use in spinal fusion in an animal model. Eli Lilly Pharmaceuticals, The lohns Hopkins Neurological Pain Research Institute, The Gordon and Marilyn Macklin Foundation funds were received to support this work. Relevant financial activities outside the submitted work: board membership, consultancy, grants/grants pending, stock/stock options, travel/ accommodations/meeting expenses and grants. Address correspondence and reprint requests to Timothy F. Witham, MD, Department of Neurosurgery, The Johns FHopkins Hospital, 600 North Wolfe St, Meyer 7-109, Baltimore, MD 21287; E-mail: [email protected] DOI: 10.1097/BRS.OOOOOOOOOOOOOl 69 Spine
M e t l i o d s . Forty-eight male New Zealand white rabbits underwent bilateral posterolateral intertransverse process arthrodesis surgery at the L5-L6 level. Animals were divided into 6 groups. Two groups were treated with autograft alone or autograft and PTH (1-34), whereas the other 4 groups were treated with low-dose rhBMP-2 alone, high-dose rhBMP-2 alone, or either dose combined with PTH (1 -34). All animals were euthanized 6 weeks after surgery. The L4-L7 spinal segment was removed and assessed using manual palpation, computed tomography (CT), and biomechanical testing. Results. CT assessments revealed fusion in 5 0 % of autograft controls, 75% of autograft PTH (1-34) animals, 87.5% in the 2 groups treated with low-dose rhBMP-2, and 100% in the 2 groups treated with high-dose rhBMP-2. CT volumetric analysis demonstrated that all groups treated with biologies had fusion masses that were on average significantly larger than those observed in the control group (P < 0.0001). Biomechanical data demonstrated no statistical difference between controls, PTH (1-34), and low-dose rhBMP-2 in any testing orientation. PTH (1-34) did not increase bending stiffness when used adjunctively with either low-dose or high-dose rhBMP-2. Conclusion. Although intermittent teriparatide administration results in increased fusion mass volume, it does not improve biomechnical stiffness over use of autograft alone. When delivered concurrently with high- and low-dose rhBMP-2, teriparatide provided no statistically significant improvement in biomechanical stiffness. K e y w o r d s : rabbit model, recombinant human bone morphogenetic protein-2, parathyroid hormone, posterolateral spinal fusion, bone graft substitute, 4-point bending stiffness, tricalcium phosphate, hydroxyapatite, pseudarthrosis, volumetric analysis.
Level of Evidence: N/A Spine 2014;39:347-355
' pinal arthrodesis, the process in which 2 or more ver|tebral segments are surgically manipulated to promote 'their fusion,' is indicated for a variety of pathological states including spinal instability secondary to trauma, infection, or neoplasm as well as intractable axial pain caused by www.spinejournai.com
347
BASIC SCIENCE
degenerative disorders.' More than 250,000 lumbar spinal fusion operations are performed annually in the United States,^ and, by far, dorsolateral intertransverse process arthrodesis (DIPA) is the most common type of fusion technique performed in the lumbar spine.** Since DIPA was first described by Watkins,' much progress has been made with respect to the instrumentation and osteoinductive agents used to promote successful arthrodesis. Although DIPA is generally considered safe and successful, the rates of nonunion according to the literature range from 5% to 35%.*"'* Specifically, failure of osseous fusion is of greater concern particularly in patients with poor or diminished bone quality resulting from osteoporosis, chronic smoking, alcohol abuse, or morbid obesity.** Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been increasingly used to enhance spinal fusion outcomes in clinical practice and has been shown to enhance spinal fusion in the New Zealand White (NZW) rabbit model.'""'-' Despite FDA approval, its use remains controversial."" Teriparatide (parathyroid hormone [PTH] [1-34]), a drug approved for osteoporosis, has recently gained interest for its potential ability to enhance bone formation because it has already been shown to enhance spinal fusion outcomes in the NZW rabbit model.-""^' However, there has been no report of the combined effects of teriparatide administered with rhBMP-2 in clinical practice or in the well-established NZW rabbit model. In the following study, we compare the efficacy of PTH (1-34) to rhBMP-2 for DIPA in the NZW rabbit model. Specifically, we aim to evaluate if the use of both PTH (1-34) and rhBMP-2 has a synergistic effect on the strength and composition of the bone fusion mass. The use of PTH (1-34) as an alternative or synergistically with rhBMP-2 may provide an important therapy for patients who are at high risk of pseudarthrosis after spinal fusion surgery.
MATERIALS AND METHODS The following study has been approved by the Johns Hopkins University Animal Care and Use Committee. Forty-eight skeletally mature (3-5 kg) male NZW rabbits were divided into 6 experimental drug groups: iliac crest autograft control, iliac crest autograft control plus PTH (1-34), low-dose rhBMP-2, high-dose rhBMP-2, low-dose rhBMP-2 plus PTH (1-34), and high-dose rhBMP-2 plus PTH (1-34). Animals were housed individually at an on-site animal facility and monitored daily. All animals were euthanized at 6 weeks after the date of surgery. Surgical Procedure Each rabbit was initially anesthetized using a solution of ketamine (35 mg/kg) and xylazine (5 mg/kg) administered intramuscularly in the hind leg. The rabbit was shaved, coated in Betadine, and draped under sterile conditions. Body temperature was maintained at 37° C using a built-in heater in the surgical table unit. Sterile gowns, gloves, caps, and masks were used by all surgical personnel. Intraoperative anesthesia was maintained using isoflurane delivered via a nose cone. Cardiorespiratory monitoring was performed throughout the procedure. 348
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PTH (l-34)/rhBMP-2 on Spinal Fusion • Lina er al
After the rabbit was placed in the prone position, a dorsal midline skin incision extending from L4-L7 was made followed by a paramedian fascial incision. An intermuscular plane was established between the multifidus and longissimus muscle layers using blunt dissection facilitating exposure of the transverse processes of L5 and L6 as well as the intertransverse membrane. An electric cauterizer was used as needed to minimize blood loss. For the control and PTH (1-34) groups, approximately 2 to 2.5 cm^ of corticocancellous bone graft was harvested from the ipsilateral iliac crest by extending the intermuscular incision inferiorly. Using an electric burr (Stryker Saber drill; Portage, MI) the L5 and L6 transverse processes were decorticated as was the lateral third of the pars of the L6 vertebra. The iliac crest bone graft was ground using rongeurs then placed in the paraspinal bed between the L5 and L6 transverse processes. For the rhBMP-2 and PTH (1-34) plus rhBMP-2 groups, 1.67 mL of synthetic bone matrix composed of 15% hydroxyapatite and 85% ß-tricalcium phosphate (MasterGraft Matrix 20-mL Kit; Medtronic) was used in place of autograft. The rhBMP-2 (Infuse Bone Graft Kit; Medtronic, Minneapolis, MN) was loaded onto a collagen vehicle 20 minutes prior to implantation at a dose of 0.080 mg per side for low-dose rhBMP-2 experimental groups and 0.656 mg per side for high-dose rhBMP-2 treatments. The fascial incision was closed with 4-0 synthetic glycolide/lactide copolymer absorbable sutures. The aforementioned procedure was repeated on the contralateral side. After closure of the dorsal midline incision, the surgical site was coated once more with Betadine.
Postsurgical Care Postoperative buprenorphine hydrochloride (0.03 mg/kg) was administered intramuscularly for pain management. In the days after surgery, rabbits were closely monitored for any sign of nerve palsy, hemiparesis, ataxia, or tremor as well as any change in overall condition. In addition, they were monitored for signs of distress or pain as evidenced by decreased or no appetite, little or no movement, or lethargy. Signs of pain were promptly treated with buprenorphine (0.01 mg/kg) intramuscularly every 12 hours for 3 doses. If there was no significant response, one of the animal resource veterinarians was consulted. Starting at postoperative day 1, animals in PTH (1-34) treatment groups and autograft groups were given daily injections of either PTH (1-34) or a control solution, respectively. The PTH (1-34) solution (Forteo, Eli Eilly Pharmaceuticals, Indianapolis, IN) was prepared in a compound sterile solution vehicle containing 40 mg/mL of mannitol and 20 mM sodium phosphate buffer in saline such that each animal received 10 |xg/kg of drug.^^ Control animals received the vehicle only. Injections were given subcutaneously in the dorsal aspect of the hind leg of the animal. Animals were euthanized after 6 weeks. The act of killing consisted of administration of initial anesthesia via ketamine and xylazine injection, followed by intracardiac injection of the appropriate amount of sodium pentobarbital solution. March 2014
BASIC SCIENCE
PTH (l-34)/rhBMP-2 on Spinal Fusion • Lina et al
Manual Palpation Once harvested, each spine was dissected so as to isolate the L4-L7 vertebral segments, removing all surrounding tnuscle and soft tissue, leaving only the joint capsule and interspinous ligaments intact (Figure 1). Manual palpation was used at the time of harvest at the level of the operative segment as well as the adjacent levels proximally and distally. The size and kinetics of each specimen was considered when determining if pseudarthrosis was present. The principal investigator was blinded and evaluated all the spines, which were graded as bilaterally fused, unilaterally fused, or not fused. Radiographical Analysis Computed tomographic (CT) scans of the harvested lumbar spines were obtained using a small animal radiation research platform developed at our institution. Two-dimensional reconstructions of the fused spine segments were generated from the raw CT data. The sagittal, coronal, and axial CT images were graded by the senior author, an experienced neurosurgeon, in a blinded fashion, as solid or not solid, based on the presence of a continuous trabecular pattern within the intertransverse fusion mass. Axial cross sections were generated to quantitatively calculate fusion mass volume. For each specimen, the region of interest was defined as any new bone formation lateral to the tangential plane corresponding to the pars of the involved vertebrae excluding any lateral portion of the transverse process that did not come into contact with the fusion mass (Figure 2). Using the ImageJ (US National Institutes of Health, Bethesda, MD) softrware, volumes were estimated on an unbiased stereology grid count system using the Volumest plugin.^^ In our analysis, each voxel of the reconstruction corresponded to a 0.2 mm-^ volume.
Biomechanical Analysis After the radiographical study, the spines were stored in a freezer at — 80" C until mechanical testing could be performed.
Figure 1. Image of a harvested high-dose rhBMP-2-treated rabbit spine (L4-L7) prior to PMMA potting for biomechanical testing with fusion present between the L5-L6 transverse processes. Arrows indicate the location of the fusion mass. rhBMP-2 indicates recombinant human bone morphogenetic protein-2. Spine
Figure 2. Axial CT image with the ROI highlighted in yellow. Osteolysis within a cortical shell of bone is noted in this high-dose rhBMP-2treated animal (arrows). rhBMP-2 indicates recombinant human bone morphogenetic protein-2; ROI, region of interest; CT, computed tomography.
Prior to testing, each spine was thawed and the transverse processes of the L4 and L7 vertebral bodies were reduced to ensure adequate motion of the L5 and L6 vertebral bodies. Each specimen was potted with poly(methyl methacrylate) (PMMA) (COE Tray Plastic Fast Set; GC America Inc., Chicago, IE) using customized aluminum box-cut casing such that both the E4-E5 and E6-E7 disk spaces were encased in cement, allowing free motion across the E5-E6 segment only. This corresponded to the E5-E6 vertebral disc being centered between tbe 2 potting blocks. Tbe PMMA was given 30 minutes to solidify sufficiently, at wbicb point the aluminum casing was removed, leaving tbe adjacent spinal segments encased in PMMA (Figure 3). The cemented spines were tben tested nondestructively using an axial mechanical tester (Bose Electroforce 3230 Series II; Bose Corporation, Eden Prairie, MN) using a 4-point bending fixture. Each specimen was tested in flexion, extension, and left and rigbt lateral bending. Tbe order of the testing orientation was randomized for each. Tbe titanium 4-point bending fixtures were adjusted to an exterior span of 100 mm (bottom) and 20 mm (top). A 10-N preload was applied prior to testing all specimens. Eacb spine underwent 7 load-unload cycles to 50 N at a rate of 5 N/s. Preliminary testing revealed adequate preconditioning of tbe specimen after tbe fifth load cycle. As a result, data from the seventb load cycle and tbe test geometry were used to calculate tbe bending stiffness, defined as El, wbere E is tbe Young's modulus and I is the second moment of inertia, in units of Nm^ of tbe specimen.-'*"^'' The specimens were repotted in cases wbere excessive motion was seen at tbe PMMA-vertebra junction at tbe adjacent segments. www.spinejournal.com
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PTH ( l-34)/rhBMP-2 on Spinal Fusion • Lina et al
Figure 3. Rabbit spine potted bilaterally in PMMA such that the L5-L6 intervertebral disc is centered between the blocks.
Statistical Methods The primary outcome variables for this study include the following: mechanical bending stiffness, bone volume measurements, and maximum axial cross-sectional area for each treatment group. Descriptive data are shown as the mean ± 1 standard error of the mean. The x" test was used to compare categorical data sets (nonfused vs. unilaterally or bilaterally fused) with intergroup differences assessed through calculation of standardized residuals. Continuous data (bending stiffness, volume, and maximum area) were compared using multiple unpaired Student t tests with Welch correction. Significances were set at P < 0.05. RESULTS During the entirety of the study, no incidence of infection was noted in any of the 6 treatment arms. All animals were observed to be eating adequately and maintaining their preoperative weight. Three rabbits were euthanized prior to the 6-week time point. Of these rabbits, 2 experienced surgical complications: 1 demonstrated extreme lethargy postoperatively and was euthanized on postoperative day 3 and the other incurred right leg paralysis, likely from sciatic nerve injury during autograft harvest. One rabbit was euthanized for an unrelated cause, toe bleeding from an inadvertent mechanical injury, per our Animal Care and Use Committee protocol. Several animals treated with high-dose rhBMP-2 were noted as having mild wound site inflammation with one A
Manual palpation
animal developing a gross subcutaneous hematoma, which was subsequently drained percutaneously. Evaluation based on CT radiographs revealed unilateral or bilateral fusion in 50% of spines in the autograft control group, 75% of spines in the PTH (l-34)-treated group, 87.5% in both low-dose rhBMP-2-treated groups, and 100% in both high-dose rhBMP-2-treated groups (Figure 4A). Manual palpation demonstrated fusion in 75% of the PTH (l-34)-treated and autograft control group with half of controls being identified as unilaterally fused (Figure 4B). Animals treated with rhBMP-2 of either low or high dosage were qualitatively assessed on CT scans as having "more solid" fusions with a greater amount of trabecular bone crossing the L5-L6 junction. Upon further dissection, several of these spines were noted as having osteolysis within the fusion mass underneath a cortical rim of bone. Heterotopic bone was noted at some adjacent levels of the spine, a finding that was particularly evident in rabbits treated with high-dose rhBMP-2. Volumetric analysis of the fusion masses (Figure 5) revealed that all groups treated with biologies demonstrated a statistically significant increase in bone volume when compared with controls (Table 1) (P < 0.0001). Flowever, adjunctive use of PTH (1-34) in addition to either dose of rhBMP-2 did not result in statistically significant increases in fusion volume when compared with experimental groups treated with rhBMP-2 alone. The maximum axial cross-sectional area data were consistent with the volumetric data (Figure 6).
CT rendering
8' No Fusion Unilateral fusion Bilateral fusion
Figure 4. Fusion assessment results: A, Manual palpation, x' ^ = 0.016. B, CT renderings, X' P = 0.011. PTH indicates parathyroid hormone; rhBMP, recombinant human bone morphogenetic protein-2; CT, computed tomography. 350
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March 2014
BASIC SCIENCE
PTH (l-34)/rhBMP-2 on Spinal Fusion • Lina et al
Volume
compared with treatment with autograft alone. Similar to volume and cross-sectional area assessments, biomechanical stiffness was not significantly enhanced when rabbits were treated with PTH (1-34) in addition to rhBMP-2 implanted locally (Table 2). However, fusion constructs in rabbits treated with high-dose rhBMP-2 were found to have significantly higher biomechanical stiffness when compared with fusion masses in rabbits treated with low-dose rhBMP-2 (P < 0.05).
u
DISCUSSION
rhBMP-2 (LO)
*
PTH (1-34) + rhBMP-2 (LO)
PTH (1-34) -1rhBMP-2 (LO)
t
t
NS
NS
NS
NS NS
44-
NS
§
NS
rhBMP-2 (LO)
44-
PTH (1-34) -H rhBMP-2 (Hl)
+
PTH (1-34) -1rhBMP-2 (HI) *
44-
*
rhBMP-2 (HI)
-H-
rhBMP-2 (HI)
44-
PTH (1-34)
PTH (1-34) * ^^^^^^1
44-
Control
Control
44-
CSA/Volume
1
In all the 6 treatment arms, the greatest bending stiffness was observed in lateral bending orientations (Figure 7). Groups treated with either low-dose or high-dose rhBMP-2 demonstrated greater stiffness than both control and PTH (l-34)-only groups. Administration of PTH (1-34) alone did not result in increased biomechanical stiffness when
-H-
Figure 5. CT volumetric analysis in cm^ averaged across each group using our ROI. Error bars indicate standard error of the mean. *P < 0.0001. PTH indicates parathyroid hormone; rhBMP, recombinant human bone morphogenetic protein-2; ROI, region of interest; CT, computed tomography.
Pseudarthrosis after lumbar fusion continues to provide a significant clinical challenge, with many lumbar fusion procedures resulting in continued mobility between incompletely fused segments.''"* Failed spinal fusion causes recurrence of pain and other neurological sequelae, which often require revision fusion, increasing hospital costs, patient morbidity, and length of treatment. As such, there has been a recent push to develop new techniques and exogenously placed compounds to augment lumbar fusion procedures, which traditionally relied on iliac crest autograft. Specifically, in the past 2 decades, there has been increased interest in the use of biological medical products to improve on traditional autologous iliac crest techniques. One such biological rhBMP-2, was initially thought to be the solution to failed fusions. Originally approved by the FDA in 2002, rhBMP-2 is a commercially produced analogue of one BMP, a growth factor thought to play a critical role in new bone formation. rhBMP-2 encourages progenitor cell recruitment and differentiation through induction of other growth factors, including vascular endothelial growth factor.^* rhBMP-2 has been shown to be just as effective or superior to iliac crest harvesting in promoting posterolateral spinal fusions in humans.^^ Unfortunately, rhBMP-2 was later found to have significant side effects leading to an FDA-issued public health notification in 2008. A recent systematic review
t
^ ^ ^ ^ 1
+
1
NS
NS
^ ^ ^ ^ ^ ^ ^ 1
Values in gray cells: P values based on volumetric analysis. Values in white cells: P values based on cross-sectional area data. *P
Spine BASIC SCIENCE
Quantitative Study of Parathyroid Hormone (1-34) and Bone Morphogenetic Protein-2 on Spinal Fusion Outcomes in a Rabbit Model of Lumbar Dorsolateral Intertransverse Process Arthrodesis loan A. Lina, BS, Varun Puvanesarajah, BS, Jason A. Liauw, MD, Sheng-fu L. Lo, MD, David R. Santiago-Dieppa, MD, Lee Hwang, MD, Annie Mao, Ali Bydon, MD, Jean-Paul Wolinsky, MD, Daniel M. Sciubba, MD, Ziya Gokaslan, MD, Christina Holmes, PhD, and Timothy F. Witham, MD
Study Design. A posterolateral rabbit spinal fusion model was used to evaluate the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) and teriparatide (PTH [1-341) used individually and in combination on spinal fusion outcomes. O b j e c t i v e . To test the efficacy of parathyroid hormone on improving spinal fusion outcomes when used with BMP-2. S u m m a r y of Background D a t a . Of the more than 250,000 spinal fusion surgical procedures performed each year, 5% to 3 5 % of these will result in pseudarthrosis. Growing controversy on the efficacy and cost of rhBMP-2 for improving spinal fusion outcomes has presented a challenge for clinicians. Research into PTH as an adjunct therapy to rhBMP-2 for spinal fusion has not yet been investigated.
From the Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD. Acknowledgment date: August 8, 2013. First revision date: December 6, 2013. Acceptance date: December 9, 2013. The device(s)/drug(s) that is/are the subject of this manuscript is/are exempt from FDA or corresponding national regulations because: They were used in an animal model where such regulations are not applicable. Current use of recombinant human bone morphogenetic protein-2 in the clinic is FDAapproved for lumbar spinal fusion surgery. This is functionally analogous to the posterolateral intertransverse arthrodesis procedure that is used In our rabbit model. On the contrary, parathyroid hormone (PTH) (1-34), although FDA-approved for the treatment of patients with osteoporosis, has been used in the literature for similar New Zealand White rabbit spinal fusion models. Concurrently, FDA regulations do not apply to this model. The manuscript includes unlabeled/investigational uses of the products/devices listed below and the status of these is disclosed in the manuscript: teriparatide is approved for use in the treatment of osteoporosis. In this study, we investigate its use in spinal fusion in an animal model. Eli Lilly Pharmaceuticals, The lohns Hopkins Neurological Pain Research Institute, The Gordon and Marilyn Macklin Foundation funds were received to support this work. Relevant financial activities outside the submitted work: board membership, consultancy, grants/grants pending, stock/stock options, travel/ accommodations/meeting expenses and grants. Address correspondence and reprint requests to Timothy F. Witham, MD, Department of Neurosurgery, The Johns FHopkins Hospital, 600 North Wolfe St, Meyer 7-109, Baltimore, MD 21287; E-mail: [email protected] DOI: 10.1097/BRS.OOOOOOOOOOOOOl 69 Spine
M e t l i o d s . Forty-eight male New Zealand white rabbits underwent bilateral posterolateral intertransverse process arthrodesis surgery at the L5-L6 level. Animals were divided into 6 groups. Two groups were treated with autograft alone or autograft and PTH (1-34), whereas the other 4 groups were treated with low-dose rhBMP-2 alone, high-dose rhBMP-2 alone, or either dose combined with PTH (1 -34). All animals were euthanized 6 weeks after surgery. The L4-L7 spinal segment was removed and assessed using manual palpation, computed tomography (CT), and biomechanical testing. Results. CT assessments revealed fusion in 5 0 % of autograft controls, 75% of autograft PTH (1-34) animals, 87.5% in the 2 groups treated with low-dose rhBMP-2, and 100% in the 2 groups treated with high-dose rhBMP-2. CT volumetric analysis demonstrated that all groups treated with biologies had fusion masses that were on average significantly larger than those observed in the control group (P < 0.0001). Biomechanical data demonstrated no statistical difference between controls, PTH (1-34), and low-dose rhBMP-2 in any testing orientation. PTH (1-34) did not increase bending stiffness when used adjunctively with either low-dose or high-dose rhBMP-2. Conclusion. Although intermittent teriparatide administration results in increased fusion mass volume, it does not improve biomechnical stiffness over use of autograft alone. When delivered concurrently with high- and low-dose rhBMP-2, teriparatide provided no statistically significant improvement in biomechanical stiffness. K e y w o r d s : rabbit model, recombinant human bone morphogenetic protein-2, parathyroid hormone, posterolateral spinal fusion, bone graft substitute, 4-point bending stiffness, tricalcium phosphate, hydroxyapatite, pseudarthrosis, volumetric analysis.
Level of Evidence: N/A Spine 2014;39:347-355
' pinal arthrodesis, the process in which 2 or more ver|tebral segments are surgically manipulated to promote 'their fusion,' is indicated for a variety of pathological states including spinal instability secondary to trauma, infection, or neoplasm as well as intractable axial pain caused by www.spinejournai.com
347
BASIC SCIENCE
degenerative disorders.' More than 250,000 lumbar spinal fusion operations are performed annually in the United States,^ and, by far, dorsolateral intertransverse process arthrodesis (DIPA) is the most common type of fusion technique performed in the lumbar spine.** Since DIPA was first described by Watkins,' much progress has been made with respect to the instrumentation and osteoinductive agents used to promote successful arthrodesis. Although DIPA is generally considered safe and successful, the rates of nonunion according to the literature range from 5% to 35%.*"'* Specifically, failure of osseous fusion is of greater concern particularly in patients with poor or diminished bone quality resulting from osteoporosis, chronic smoking, alcohol abuse, or morbid obesity.** Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been increasingly used to enhance spinal fusion outcomes in clinical practice and has been shown to enhance spinal fusion in the New Zealand White (NZW) rabbit model.'""'-' Despite FDA approval, its use remains controversial."" Teriparatide (parathyroid hormone [PTH] [1-34]), a drug approved for osteoporosis, has recently gained interest for its potential ability to enhance bone formation because it has already been shown to enhance spinal fusion outcomes in the NZW rabbit model.-""^' However, there has been no report of the combined effects of teriparatide administered with rhBMP-2 in clinical practice or in the well-established NZW rabbit model. In the following study, we compare the efficacy of PTH (1-34) to rhBMP-2 for DIPA in the NZW rabbit model. Specifically, we aim to evaluate if the use of both PTH (1-34) and rhBMP-2 has a synergistic effect on the strength and composition of the bone fusion mass. The use of PTH (1-34) as an alternative or synergistically with rhBMP-2 may provide an important therapy for patients who are at high risk of pseudarthrosis after spinal fusion surgery.
MATERIALS AND METHODS The following study has been approved by the Johns Hopkins University Animal Care and Use Committee. Forty-eight skeletally mature (3-5 kg) male NZW rabbits were divided into 6 experimental drug groups: iliac crest autograft control, iliac crest autograft control plus PTH (1-34), low-dose rhBMP-2, high-dose rhBMP-2, low-dose rhBMP-2 plus PTH (1-34), and high-dose rhBMP-2 plus PTH (1-34). Animals were housed individually at an on-site animal facility and monitored daily. All animals were euthanized at 6 weeks after the date of surgery. Surgical Procedure Each rabbit was initially anesthetized using a solution of ketamine (35 mg/kg) and xylazine (5 mg/kg) administered intramuscularly in the hind leg. The rabbit was shaved, coated in Betadine, and draped under sterile conditions. Body temperature was maintained at 37° C using a built-in heater in the surgical table unit. Sterile gowns, gloves, caps, and masks were used by all surgical personnel. Intraoperative anesthesia was maintained using isoflurane delivered via a nose cone. Cardiorespiratory monitoring was performed throughout the procedure. 348
www.spinejournal.com
PTH (l-34)/rhBMP-2 on Spinal Fusion • Lina er al
After the rabbit was placed in the prone position, a dorsal midline skin incision extending from L4-L7 was made followed by a paramedian fascial incision. An intermuscular plane was established between the multifidus and longissimus muscle layers using blunt dissection facilitating exposure of the transverse processes of L5 and L6 as well as the intertransverse membrane. An electric cauterizer was used as needed to minimize blood loss. For the control and PTH (1-34) groups, approximately 2 to 2.5 cm^ of corticocancellous bone graft was harvested from the ipsilateral iliac crest by extending the intermuscular incision inferiorly. Using an electric burr (Stryker Saber drill; Portage, MI) the L5 and L6 transverse processes were decorticated as was the lateral third of the pars of the L6 vertebra. The iliac crest bone graft was ground using rongeurs then placed in the paraspinal bed between the L5 and L6 transverse processes. For the rhBMP-2 and PTH (1-34) plus rhBMP-2 groups, 1.67 mL of synthetic bone matrix composed of 15% hydroxyapatite and 85% ß-tricalcium phosphate (MasterGraft Matrix 20-mL Kit; Medtronic) was used in place of autograft. The rhBMP-2 (Infuse Bone Graft Kit; Medtronic, Minneapolis, MN) was loaded onto a collagen vehicle 20 minutes prior to implantation at a dose of 0.080 mg per side for low-dose rhBMP-2 experimental groups and 0.656 mg per side for high-dose rhBMP-2 treatments. The fascial incision was closed with 4-0 synthetic glycolide/lactide copolymer absorbable sutures. The aforementioned procedure was repeated on the contralateral side. After closure of the dorsal midline incision, the surgical site was coated once more with Betadine.
Postsurgical Care Postoperative buprenorphine hydrochloride (0.03 mg/kg) was administered intramuscularly for pain management. In the days after surgery, rabbits were closely monitored for any sign of nerve palsy, hemiparesis, ataxia, or tremor as well as any change in overall condition. In addition, they were monitored for signs of distress or pain as evidenced by decreased or no appetite, little or no movement, or lethargy. Signs of pain were promptly treated with buprenorphine (0.01 mg/kg) intramuscularly every 12 hours for 3 doses. If there was no significant response, one of the animal resource veterinarians was consulted. Starting at postoperative day 1, animals in PTH (1-34) treatment groups and autograft groups were given daily injections of either PTH (1-34) or a control solution, respectively. The PTH (1-34) solution (Forteo, Eli Eilly Pharmaceuticals, Indianapolis, IN) was prepared in a compound sterile solution vehicle containing 40 mg/mL of mannitol and 20 mM sodium phosphate buffer in saline such that each animal received 10 |xg/kg of drug.^^ Control animals received the vehicle only. Injections were given subcutaneously in the dorsal aspect of the hind leg of the animal. Animals were euthanized after 6 weeks. The act of killing consisted of administration of initial anesthesia via ketamine and xylazine injection, followed by intracardiac injection of the appropriate amount of sodium pentobarbital solution. March 2014
BASIC SCIENCE
PTH (l-34)/rhBMP-2 on Spinal Fusion • Lina et al
Manual Palpation Once harvested, each spine was dissected so as to isolate the L4-L7 vertebral segments, removing all surrounding tnuscle and soft tissue, leaving only the joint capsule and interspinous ligaments intact (Figure 1). Manual palpation was used at the time of harvest at the level of the operative segment as well as the adjacent levels proximally and distally. The size and kinetics of each specimen was considered when determining if pseudarthrosis was present. The principal investigator was blinded and evaluated all the spines, which were graded as bilaterally fused, unilaterally fused, or not fused. Radiographical Analysis Computed tomographic (CT) scans of the harvested lumbar spines were obtained using a small animal radiation research platform developed at our institution. Two-dimensional reconstructions of the fused spine segments were generated from the raw CT data. The sagittal, coronal, and axial CT images were graded by the senior author, an experienced neurosurgeon, in a blinded fashion, as solid or not solid, based on the presence of a continuous trabecular pattern within the intertransverse fusion mass. Axial cross sections were generated to quantitatively calculate fusion mass volume. For each specimen, the region of interest was defined as any new bone formation lateral to the tangential plane corresponding to the pars of the involved vertebrae excluding any lateral portion of the transverse process that did not come into contact with the fusion mass (Figure 2). Using the ImageJ (US National Institutes of Health, Bethesda, MD) softrware, volumes were estimated on an unbiased stereology grid count system using the Volumest plugin.^^ In our analysis, each voxel of the reconstruction corresponded to a 0.2 mm-^ volume.
Biomechanical Analysis After the radiographical study, the spines were stored in a freezer at — 80" C until mechanical testing could be performed.
Figure 1. Image of a harvested high-dose rhBMP-2-treated rabbit spine (L4-L7) prior to PMMA potting for biomechanical testing with fusion present between the L5-L6 transverse processes. Arrows indicate the location of the fusion mass. rhBMP-2 indicates recombinant human bone morphogenetic protein-2. Spine
Figure 2. Axial CT image with the ROI highlighted in yellow. Osteolysis within a cortical shell of bone is noted in this high-dose rhBMP-2treated animal (arrows). rhBMP-2 indicates recombinant human bone morphogenetic protein-2; ROI, region of interest; CT, computed tomography.
Prior to testing, each spine was thawed and the transverse processes of the L4 and L7 vertebral bodies were reduced to ensure adequate motion of the L5 and L6 vertebral bodies. Each specimen was potted with poly(methyl methacrylate) (PMMA) (COE Tray Plastic Fast Set; GC America Inc., Chicago, IE) using customized aluminum box-cut casing such that both the E4-E5 and E6-E7 disk spaces were encased in cement, allowing free motion across the E5-E6 segment only. This corresponded to the E5-E6 vertebral disc being centered between tbe 2 potting blocks. Tbe PMMA was given 30 minutes to solidify sufficiently, at wbicb point the aluminum casing was removed, leaving tbe adjacent spinal segments encased in PMMA (Figure 3). The cemented spines were tben tested nondestructively using an axial mechanical tester (Bose Electroforce 3230 Series II; Bose Corporation, Eden Prairie, MN) using a 4-point bending fixture. Each specimen was tested in flexion, extension, and left and rigbt lateral bending. Tbe order of the testing orientation was randomized for each. Tbe titanium 4-point bending fixtures were adjusted to an exterior span of 100 mm (bottom) and 20 mm (top). A 10-N preload was applied prior to testing all specimens. Eacb spine underwent 7 load-unload cycles to 50 N at a rate of 5 N/s. Preliminary testing revealed adequate preconditioning of tbe specimen after tbe fifth load cycle. As a result, data from the seventb load cycle and tbe test geometry were used to calculate tbe bending stiffness, defined as El, wbere E is tbe Young's modulus and I is the second moment of inertia, in units of Nm^ of tbe specimen.-'*"^'' The specimens were repotted in cases wbere excessive motion was seen at tbe PMMA-vertebra junction at tbe adjacent segments. www.spinejournal.com
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PTH ( l-34)/rhBMP-2 on Spinal Fusion • Lina et al
Figure 3. Rabbit spine potted bilaterally in PMMA such that the L5-L6 intervertebral disc is centered between the blocks.
Statistical Methods The primary outcome variables for this study include the following: mechanical bending stiffness, bone volume measurements, and maximum axial cross-sectional area for each treatment group. Descriptive data are shown as the mean ± 1 standard error of the mean. The x" test was used to compare categorical data sets (nonfused vs. unilaterally or bilaterally fused) with intergroup differences assessed through calculation of standardized residuals. Continuous data (bending stiffness, volume, and maximum area) were compared using multiple unpaired Student t tests with Welch correction. Significances were set at P < 0.05. RESULTS During the entirety of the study, no incidence of infection was noted in any of the 6 treatment arms. All animals were observed to be eating adequately and maintaining their preoperative weight. Three rabbits were euthanized prior to the 6-week time point. Of these rabbits, 2 experienced surgical complications: 1 demonstrated extreme lethargy postoperatively and was euthanized on postoperative day 3 and the other incurred right leg paralysis, likely from sciatic nerve injury during autograft harvest. One rabbit was euthanized for an unrelated cause, toe bleeding from an inadvertent mechanical injury, per our Animal Care and Use Committee protocol. Several animals treated with high-dose rhBMP-2 were noted as having mild wound site inflammation with one A
Manual palpation
animal developing a gross subcutaneous hematoma, which was subsequently drained percutaneously. Evaluation based on CT radiographs revealed unilateral or bilateral fusion in 50% of spines in the autograft control group, 75% of spines in the PTH (l-34)-treated group, 87.5% in both low-dose rhBMP-2-treated groups, and 100% in both high-dose rhBMP-2-treated groups (Figure 4A). Manual palpation demonstrated fusion in 75% of the PTH (l-34)-treated and autograft control group with half of controls being identified as unilaterally fused (Figure 4B). Animals treated with rhBMP-2 of either low or high dosage were qualitatively assessed on CT scans as having "more solid" fusions with a greater amount of trabecular bone crossing the L5-L6 junction. Upon further dissection, several of these spines were noted as having osteolysis within the fusion mass underneath a cortical rim of bone. Heterotopic bone was noted at some adjacent levels of the spine, a finding that was particularly evident in rabbits treated with high-dose rhBMP-2. Volumetric analysis of the fusion masses (Figure 5) revealed that all groups treated with biologies demonstrated a statistically significant increase in bone volume when compared with controls (Table 1) (P < 0.0001). Flowever, adjunctive use of PTH (1-34) in addition to either dose of rhBMP-2 did not result in statistically significant increases in fusion volume when compared with experimental groups treated with rhBMP-2 alone. The maximum axial cross-sectional area data were consistent with the volumetric data (Figure 6).
CT rendering
8' No Fusion Unilateral fusion Bilateral fusion
Figure 4. Fusion assessment results: A, Manual palpation, x' ^ = 0.016. B, CT renderings, X' P = 0.011. PTH indicates parathyroid hormone; rhBMP, recombinant human bone morphogenetic protein-2; CT, computed tomography. 350
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March 2014
BASIC SCIENCE
PTH (l-34)/rhBMP-2 on Spinal Fusion • Lina et al
Volume
compared with treatment with autograft alone. Similar to volume and cross-sectional area assessments, biomechanical stiffness was not significantly enhanced when rabbits were treated with PTH (1-34) in addition to rhBMP-2 implanted locally (Table 2). However, fusion constructs in rabbits treated with high-dose rhBMP-2 were found to have significantly higher biomechanical stiffness when compared with fusion masses in rabbits treated with low-dose rhBMP-2 (P < 0.05).
u
DISCUSSION
rhBMP-2 (LO)
*
PTH (1-34) + rhBMP-2 (LO)
PTH (1-34) -1rhBMP-2 (LO)
t
t
NS
NS
NS
NS NS
44-
NS
§
NS
rhBMP-2 (LO)
44-
PTH (1-34) -H rhBMP-2 (Hl)
+
PTH (1-34) -1rhBMP-2 (HI) *
44-
*
rhBMP-2 (HI)
-H-
rhBMP-2 (HI)
44-
PTH (1-34)
PTH (1-34) * ^^^^^^1
44-
Control
Control
44-
CSA/Volume
1
In all the 6 treatment arms, the greatest bending stiffness was observed in lateral bending orientations (Figure 7). Groups treated with either low-dose or high-dose rhBMP-2 demonstrated greater stiffness than both control and PTH (l-34)-only groups. Administration of PTH (1-34) alone did not result in increased biomechanical stiffness when
-H-
Figure 5. CT volumetric analysis in cm^ averaged across each group using our ROI. Error bars indicate standard error of the mean. *P < 0.0001. PTH indicates parathyroid hormone; rhBMP, recombinant human bone morphogenetic protein-2; ROI, region of interest; CT, computed tomography.
Pseudarthrosis after lumbar fusion continues to provide a significant clinical challenge, with many lumbar fusion procedures resulting in continued mobility between incompletely fused segments.''"* Failed spinal fusion causes recurrence of pain and other neurological sequelae, which often require revision fusion, increasing hospital costs, patient morbidity, and length of treatment. As such, there has been a recent push to develop new techniques and exogenously placed compounds to augment lumbar fusion procedures, which traditionally relied on iliac crest autograft. Specifically, in the past 2 decades, there has been increased interest in the use of biological medical products to improve on traditional autologous iliac crest techniques. One such biological rhBMP-2, was initially thought to be the solution to failed fusions. Originally approved by the FDA in 2002, rhBMP-2 is a commercially produced analogue of one BMP, a growth factor thought to play a critical role in new bone formation. rhBMP-2 encourages progenitor cell recruitment and differentiation through induction of other growth factors, including vascular endothelial growth factor.^* rhBMP-2 has been shown to be just as effective or superior to iliac crest harvesting in promoting posterolateral spinal fusions in humans.^^ Unfortunately, rhBMP-2 was later found to have significant side effects leading to an FDA-issued public health notification in 2008. A recent systematic review
t
^ ^ ^ ^ 1
+
1
NS
NS
^ ^ ^ ^ ^ ^ ^ 1
Values in gray cells: P values based on volumetric analysis. Values in white cells: P values based on cross-sectional area data. *P
